Alzheimer's disease (AD) is a progressive disease known generally as senile dementia. Broadly speaking, the disease falls into two categories, namely late onset and early onset. Late onset, which occurs in old age (65+ years), may be caused by the natural atrophy of the brain occurring at a faster rate and to a more severe degree than normal. Early onset AD is much more infrequent but shows a pathologically identical dementia with diffuse brain atrophy which develops well before the senile period, i.e., between the ages of 35 and 60 years. There is evidence that one form of this type of AD shows a tendency to run in families and is therefore known as familial Alzheimer's disease (FAD).
In both types of AD the pathology is the same but the abnormalities tend to be more severe and more widespread in cases beginning at an earlier age. The disease is characterized by two types of lesions in the brain: senile plaques and neurofibrillary tangles.
Senile plaques are areas of disorganized neuropil up to 150 mm across with extra cellular amyloid deposits at the center. Neurofibrillary tangles are intracellular deposits of amyloid protein consisting of two filaments twisted about each other in pairs.
The major protein subunit, B-amyloid protein (also referred to in the art as amyloid B protein (ABP) and A4) of the amyloid filaments of both the neurofibrillary tangle and the senile plaque, is a highly aggregating small polypeptide of approximate relative molecular mass 4,500. This protein is a cleavage product of a much larger precursor protein called amyloid precursor protein (APP).
The sequence of the deposited B-amyloid protein in particular brain regions-is one of the main pathologic characteristics of AD. The β-amyloid protein is an approximately 4 kD protein (39 to 42 amino acids) which is derived, as an internal cleavage product, from one or more isoforms of a larger APP. There are at least five distinct isoforms of APP: 563, 695, 714, 751, and 770 amino acids, respectively (Wirak et al. (1991) Science 253:323). These isoforms of APP are generated by alternative splicing of primary transcripts of the APP gene, which is located on human chromosome 21. It is known that the APP isoforms are glycosylated transmembrane proteins (Goldgaber et al. (1987) Science 235:877), and that two of the isoforms APP751 and APP770, have a protease inhibitor domain that is homologous to the Kunitz type of serine protease inhibitors. The β-amyloid protein segment comprises approximately half of the transmembrane domain and approximately the first 28 amino acids of the extra cellular domain of an APP isoform.
Proteolytic processing of APP in vivo is a normal physiological process. Carboxy-terminal truncated forms of APP695, APP751, and APP770 are present in brain and cerebrospinal fluid (Palmert et al. (1989) Proc. Natl. Acad. Sci. (U.S.A.) 86:6338; Weidemann et al. (1989) Cell 57:115) and result from cleavage of the APP isoform at a constitutive cleavage site within the β-amyloid protein peptide domain of an APP isoform (Esch et al. (1990) Science 248:1122). Normal proteolytic cleavage at the constitutive cleavage site yields a large (approximately 100 kD) soluble, N-terminal fragment that contains the protease inhibitor domain in some isoforms, and a 9 kD membrane-bound, C-terminal fragment that includes most of the β-amyloid protein domain.
Generation of pathogenic β-amyloid protein appears to be the result of aberrant proteolytic processing of APP, such that normal cleavage at the constitutive site within the β-amyloid protein domain does not occur, but rather cleavage occurs at two specific sites which flank the β-amyloid protein domain. One of these aberrant cleavage sites is in the transmembrane domain and the other aberrant cleavage site is located approximately at the N-terminus of the first 28 amino acids of the extra cellular domain. Such aberrant proteolytic cleavage produces the β-amyloid protein polypeptide which is prone to forming dense amyloidogenic aggregates that are resistant to proteolytic degradation and removal. The resultant β-amyloid protein aggregates presumably are involved in the formation of the abundant amyloid plaques and cerebrovascular amyloid that are the neuropathological hallmarks of AD. However, the exact aberrant cleavage sites are not always precise; β-amyloid molecules isolated from the brain of a patient with AD show N- and C-terminal heterogeneity. Therefore, the aberrant cleavage pathway may involve either sequence-specific proteolysis followed by exopeptidase activity (creating end-heterogeneity), or may not be sequence-specific.
The APP gene is known to be located on human chromosome 21. A locus segregating with FAD has been mapped to chromosome 21 (St. George Hyslop et al. (1987) Science 235:885) close to the APP gene. Recombinants between the APP gene and the AD locus have been previously reported (Schellenberg et al. (1988) Science 241:1507). The data appeared to exclude the APP gene as the site of any mutation that might cause FAD (Van Broekhoven et al. (1987) Nature 329:153; Tanzi et al. (1987) Nature 329:156).
Recombinant DNA technology provides several techniques for analyzing genes to locate possible mutations. For example, the polymerase chain reaction (John Bell (1989) Immunology Today 10:351-355) may be used to amplify specific sequences using intronic primers, which can then be analyzed by direct sequencing.
Using such techniques, a single base substitution, a C to T transition at base pair 2149, has been found in part of the sequence of the APP gene in some cases of FAD. This base pair transition leads to an amino acid substitution, i.e., valine to isoleucine at amino acid 717 (APP770) (see Yoshikai et al. (1990) Gene 87:257), close to the C-terminus of the β-amyloid protein. This suggests that some cases of AD are caused by mutation in the APP gene, specifically mutations that change codon 717 such that it encodes an amino acid other than valine.
A second APP allelic variant wherein glycine is substituted for valine at codon 717 is now identified, and is so closely linked to the AD phenotype as to indicate that allelic variants at codon 717 of the APP gene, particularly those encoding an amino acid other than valine, and more particularly those encoding an isolcucine, glycine, or phenylalanine, are pathogenic and/or pathognomonic alleles (Chartier-Harlin et al. (1991) Nature 353:844).
Proteolysis on either side of the β-amyloid protein region of APP may be enhanced or qualitatively altered by the specific mutations at codon 717, increasing the rate of β-amyloid deposition and aggregation. Such codon 717 mutations may increase β-amyloid formation by providing a poorer substrate for the main proteolytic pathway (cleavage at the constitutive site) or a better substrate for a competing, alternative cleavage pathway (at aberrant cleavage sites).
There are several disease states which give rise to progressive intellectual deterioration closely resembling the dementia associated with AD for which treatment is available. A further diagnostic test for AD would therefore provide a valuable tool in the diagnosis and treatment of these other conditions, by way of being able to exclude AD.
Also important is the development of experimental models of AD that can be used to define further the underlying biochemical events involved in AD pathogenesis. Such models could be employed to screen for agents that alter the degenerative course of AD. For example, a model system of AD could be used to screen for environmental factors that induce or accelerate the pathogenesis of AD. In contradistinction, an experimental model could be used to screen for agents that inhibit, prevent, or reverse the progression of AD. Such models could be employed to develop pharmaceuticals that are effective in preventing, arresting, or reversing AD.
The present invention provides the discovery of additional heretofore unknown mutations in β-amyloid protein. These mutations can be utilized advantageously to detect, treat and screen in subjects and model systems.